Primary cultures of adult rat hepatocytes were incubated (6 to 96 hr) with 50 to 150 mmol/L ethanol, 0.5 mmoUL linoleate, 0.5 mmoUL palmitate, 0.5 mmol/L 4-methylpyrazole, 0 to 25 Fmol/L vitamin E phosphate or selected combinations of these agents. Agent-dependent changes in liver cell viability (AST release and reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and function (phospholipid peroxidation, hydrolysis, biosynthesis and triacylglycerol biosynthesis) were determined. The influence of ethanol on liver cell function and viability was dose and incubation time dependent. Short periods (24 hr or less) of exposure to 100 mmol/L ethanol increased liver cell triacylglycerol biosynthesis and phospholipid hydrolysis, peroxidation and biosynthesis without altering cell viability. However, longer periods (72 hr or more) of exposure to 100 or 150 mmol/L ethanol resulted in significant reductions (30% to 50%) in cell viability, function and phosphatidylcholine biosynthesis and content. The ethanol-dependent decreases in cell function and viability were potentiated by linoleate and reduced by vitamin E phosphate, palmitate and 4-methylpyrazole. These results suggest that ethanol-induced liver cell injury in uitro is not a result of ethanol per se, but factors such as acetaldehyde or oxyradicals produced as a consequence of ethanol metabolism. Therefore the incubation of cultured hepatocytes with ethanol may be an appropriate model in uitro for determining the mechanisms by which ethanol intake disrupts liver cell function in uiuo. (HEPATOLOGY 1994;19:174-182.) Alcohol-dependent liver disease (ALD) is the most common form of liver disease in the western world. ALD is also the major cause of chronic medical illness and death associated with alcohol abuse (1). Despite the frequency of ethanol-dependent liver injury, understanding of the mechanisms by which ethanol ingestion results in ALD is limited. This lack of knowledge is